Cancer is the second leading cause of death next to heart attacks in the United States. There has been important progress in the development of new therapies in the treatment of this devastating disease. Much of the progress is due to better understanding of cell proliferation in both normal cells and cancerous cells.
Normal cells proliferate as a result of the highly controlled activation of growth factor receptors by their respective ligands. Examples of such receptors are the growth factor receptor tyrosine kinases.
Cancer cells also proliferate as a result of DNA mutations or loss of tumor suppressor gene function in normal cells. These genetic alterations cause many new protein products, such as overexpression of tumor-associated growth factors or chemokines or receptors, that can stimulate other cells (e.g., endothelial cells) to proliferate and form the new blood vessels within the tumor for continued growth and promotion of metastasis.
Some examples of growth factor receptors found on non-tumor cells that support tumor growth and, in certain circumstances, on the surface of tumor cells themselves involve vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFR), insulin-like growth factor receptors (IGFR), nerve growth factor receptors (NGFR), and fibroblast growth factor receptors (FGF).
During embryonic development, hematopoietic and early endothelial cells (angioblasts) originate from a common precursor cell known as hemagioblast. Because of their common cellular origin, several signaling pathways are shared by both hematopoietic and vascular cells. One such pathway is the VEGFR signaling pathway. VEGF receptors (VEGFR) include VEGFR1 (otherwise known as FLT-1), which was sequenced by Shibuya M. et al., Oncogene 5:519-524 (1990) and VEGFR2 (otherwise known as KDR or FLK-1), described in Terman et al., Oncogene 6:1677-1683 (1991); and sequenced by Matthews W. et al., Proc. Natl. Acad. Sci. USA 88:9026-9030 (1991).
Unless otherwise stated or clearly inferred otherwise by context, this specification will follow the customary literature nomenclature of VEGF receptors. KDR will be referred to as the human form of VEGFR2. FLK-1 will be referred to as the murine homolog of VEGFR2. FLT-1 is different from, but related to, the KDR/FLK-1 receptor.
VEGFR binds to both VEGFR1 and VEGFR2, exherting proliferative and migratory effects on endothelial and hematopoietic cells. VEGFR2 was thought to be exclusively expressed by endothelial cells. Recently, however, VEGFR2 has been shown to be present on a subset of multi-potent hematopoietic stem cells (Ziegler et al., Science 285(5433):1553-8 (1999)). Several studies have revealed that certain leukemic cells also expressed VEGFR1 and VEGFR2 (Fiedler et al., Blood 89(6):1870-5 (1997)).
The two primary signaling tyrosine kinase receptors that mediate the various biological effects of VEGF are VEGFR2 and VEGFR1. Although the binding affinity of VEGFR1 to VEGF is very high, with Kd values of 10-70 pM (Klagsbrun et al., Cytokine Growth Factor Rev 7(3):259-70 (1996)), most studies have shown that VEGFR2 is the critical receptor for transmitting cellular signals for the proliferation and differentiation of endothelial cells (Ortega et al., Am J Pathol 151(5):1215-24 (1997)). VEGFR1 appears to be more important for vascular remodeling. The relative significance of VEGF receptors in the regulation of vasculogenesis and angiogenesis has been established in studies in which the VEGFR2 and VEGFR1 genes were disrupted in murine embryonic stem cells by homologous recombination. Mice deficient in VEGFR2 had drastic defects in vasculogenesis, angiogenesis, and hematopoiesis (Shalaby et al., Nature 376(6535):62-6 (1995)). In contrast, VEGFR1 knockout mice developed abnormal vascular channels, suggesting a role for this receptor in the regulation of cellular interactions and vascular stabilization (Fong et al., Nature 376(6535):66-70 (1995)).
Inhibition of angiogenesis through disruption of VEGFR2 signaling results in inhibition of growth and metastasis of solid tumors. For example, neutralizing monoclonal antibody (MoAb) to murine VEGFR2 inhibited tumor invasion in murine models (Skobe et al., Nat Med 3(11):1222-7 (1997) and Prewett et al., Cancer Res 59(20):5209-18 (1999)). Furthermore, glioblastoma growth was inhibited in mice dominant-negative for VEGFR2 (Millauer et al., Nature 367(6463):576-9 (1994)). Such inhibition of tumor growth is attributed to the inhibition of angiogenesis, effectively limiting the blood supply of the tumor.
Leukemias originate from hematopoietic stem cells at different stages of their maturation and differentiation. It is now well established that acute leukemias originate from immature hematopoietic stem cells that have the capacity to undergo self-renewal, whereas certain less aggressive leukemias such as chronic leukemias seem to originate from the more mature committed hematopoietic progenitor cells.
Several studies have shown that VEGF is almost invariably expressed by all established leukemic cell lines as well as freshly isolated human leukemias, including the well studied HL-60 leukemic cell line (Fiedler et al., Blood 89(6):1870-5 (1997), Bellamy et al., Cancer Res 59(3):728-33 (1999)). Using RT-PCR, several studies have shown that VEGFR-2, and VEGFR-1 are only expressed by certain human leukemias (Fiedler et al., Blood 89(6):1870-5 (1997), Bellamy et al., Cancer Res 59(3):728-33 (1999)). However, none of these studies have shown whether expression of VEGF is associated with any parallel surface VEGFR2/VEGFR1 expression or functional response.
Bone marrow (BM)-derived cells (BMDCs) can contribute to malignant conversion (Coussens et al., “MMP-9 Supplied by Bone Marrow-derived Cells Contributes to Skin Carcinogenesis,” Cell 103:481-490 (2000)), tumor vascularization (Lyden et al., “Impaired Recruitment of Bone-marrow-derived Endothelial and Hematopoietic Precursor Cells Blocks Tumor Angiogenesis and Growth,” Nat. Med. 7:1194-1201 (2001) and Autiero et al., “Placental Growth Factor and its Receptor, Vascular Endothelial Growth Factor Receptor-1: Novel Targets for Stimulation of Ischemic Tissue Revascularization and Inhibition of Angiogenic and Inflammatory Disorders,” Journal of Thromb. Haemost. 1:1356-1370 (2003)), and neoplastic cell migration (Neson et al., “Lymphocyte-facilitated Tumor Cell Adhesion to Endothelial Cells: the Role of High Affinity Leukocyte Integrins,” Pathology 35:50-55 (2003)). A population of hematopoietic progenitor cells (HPCs) expressing vascular endothelial growth factor receptor 1 (VEGFR1) whose stem cells reside in specific niche-dependent regions in the BM have previously been identified. In neoangiogenesis, this normally small population of less than 0.01% total BM cells proliferate and mobilize to the peripheral circulation along with BM-derived VEGFR2+ endothelial progenitors (EPCs), both essential for the vascularization and growth of primary tumors (Lyden et al., “Impaired Recruitment of Bone-marrow-derived Endothelial and Hematopoietic Precursor Cells Blocks Tumor Angiogenesis and Growth,” Nat. Med. 7:1194-1201 (2001) and Hattori et al., “Placental Growth Factor Reconstitutes Hematopoiesis by Recruiting VEGFR1(+) Stem Cells from Bone-marrow Microenvironment,” Nat. Med. 8:841-9 (2002)). These VEGFR1+ cells, which are of myelomonocytic origin, localize to perivascular sites in the tumor bed and play a supportive and stabilizing role for newly formed vessels (Lyden et al., “Impaired Recruitment of Bone-marrow-derived Endothelial and Hematopoietic Precursor Cells Blocks Tumor Angiogenesis and Growth,” Nat. Med. 7:1194-1201 (2001)). These and other tumor-associated cells have been found to enhance primary tumor growth and promote tumor spread, yet their precise contribution to metastasis is currently unclear (Pollard, “Tumor-educated Macrophages Promote Tumor Progression and Metastasis,” Nat. Rev. Cancer. 4:71-78 (2004) and Hiratsuka et al., “MMP9 Induction by Vascular Endothelial Growth Factor Receptor-1 is Involved in Lung-specific Metastasis,” Cancer Cell. 2:289-300 (2002)).
The present invention is directed to utilizing these phenomena in monitoring and treating cancer and metastasis.